Sexual development dysgenesis in interspecific hybrids of Medaka fish.

Fish are amongst vertebrates the group with the highest diversity of known sex-determining genes. Particularly, the genus Oryzias is a suitable taxon to understand how different sex determination genetic networks evolved in closely related species. Two closely related species, O. latipes and O. curvinotus, do not only share the same XX/XY sex chromosome system, but also the same male sex-determining gene, dmrt1bY. We performed whole mRNA transcriptomes and morphology analyses of the gonads of hybrids resulting from reciprocal crosses between O. latipes and O. curvinotus. XY male hybrids, presenting meiotic arrest and no production of sperm were sterile, and about 30% of the XY hybrids underwent male-to-female sex reversal. Both XX and XY hybrid females exhibited reduced fertility and developed ovotestis while aging. Transcriptome data showed that male-related genes are upregulated in the XX and XY female hybrids. The transcriptomes of both types of female and of the male gonads are characterized by upregulation of meiosis and germ cell differentiation genes. Differences in the parental species in the downstream pathways of sexual development could explain sex reversal, sterility, and the development of intersex gonads in the hybrids. We hypothesize that male-to-female sex reversal may be connected to a different development time between species at which dmrt1bY expression starts. Our results provide molecular clues for the proximate mechanisms of hybrid incompatibility and Haldane's rule.

An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid.

CRISPR-Cas9 technology is a very efficient functional analysis tool and has been developed in several insects to edit their genome through injection of eggs with guide RNAs targeting coding sequences of genes of interest. However, its implementation in aphids is more challenging. Aphids are major pests of crops worldwide that alternate during their life cycle between clonality and sexual reproduction. The production of eggs after mating of sexual individuals is a single yearly event and is necessarily triggered by a photoperiod decrease. Fertilized eggs then experience an obligate 3-month diapause period before hatching as new clonal colonies. Taking into consideration these particularities, we developed in the pea aphid Acyrthosiphon pisum a step-by-step protocol of targeted mutagenesis based on the microinjection within fertilized eggs of CRISPR-Cas9 components designed for the editing of a cuticular protein gene (stylin-01). This protocol includes the following steps: i) the photoperiod-triggered induction of sexual morphs (2 months), ii) the mating and egg collection step (2 weeks), iii) egg microinjection and melanization, iv) the 3-month obligate diapause, v) the hatching of new lineages from injected eggs (2 weeks) and vi) the maintenance of stable lineages (2 weeks). Overall, this 7-month long procedure was applied to three different crosses in order to estimate the impact of the choice of the genetic combination on egg production dynamics by females as well as hatching rates after diapause. Mutation rates within eggs before diapause were estimated at 70-80%. The hatching rate of injected eggs following diapause ranged from 1 to 11% depending on the cross and finally a total of 17 stable lineages were obtained and maintained clonally. Out of these, 6 lineages were mutated at the defined sgRNAs target sites within stylin-01 coding sequence, either at the two alleles (2 lineages) or at one allele (4 lineages). The final germline transmission rate of the mutations was thus around 35%. Our protocol of an efficient targeted mutagenesis opens the avenue for functional studies through genome editing in aphids.

Expression of the male determining gene dmrt1bY and its autosomal coorthologue dmrt1a in medaka.

Dmrt1bY (aka dmy) has been identified as the master male sex-determining gene of medaka by positional cloning and genetic evidence. How this gene on the molecular level determines the differentiation of the gonad primordium towards a testis, however, is unknown. To better understand the roles of dmrt1bY and its autosomal counterpart dmrt1a in the process of sex determination, a detailed expression analysis by quantitative real-time RT-PCR was performed showing non-organ-specific expression of dmrt1bY during embryogenesis and low expression in adult testes. Surprisingly a high expression of dmrt1bY was noted in the spleen of males. Dmrt1a is exclusively expressed in adult testes, 50 times higher than dmrt1bY. Dmrt1bY mRNA and protein have short half-lives and the GFP fusion protein products of both dmrt1 genes are localized to the nucleus. These features are consistent with the suggestion that Dmrt1 proteins might function as DNA-binding transcription factors.

Structural and functional evidences for a type 1 TGF-beta sensu stricto receptor in the lophotrochozoan Crassostrea gigas suggest conserved molecular mechanisms controlling mesodermal patterning across bilateria.

The transforming growth factor beta (TGFbeta) superfamily includes bone morphogenetic proteins, activins and TGF-betasensu stricto (s.s.). These ligands have been shown to play a key role in numerous biological processes including early embryonic development and immune regulation. They transduce their signal through a hetromeric complex of type I and type II receptors. Such receptors have been identified in ecdysozoans but none have been found as yet in the other major protostomal clade, the lophotrochozoans. Here, we report the identification of the first lophotrochozoan TGFbetas.s. type I receptor (Cg-TGFbetaRI) from the mollusk Crassostrea gigas. The phylogenetic and structural analyses as well as the expression pattern during early development suggest Cg-TGFbetaRI to belong to the TGFbetas.s./activin type I receptor clade and functional studies corroborate these deductions. The use of the zebrafish embryo as a reporter organism reveals that either Cg-TGFbetaRI or its dominant negative acting truncated form, when overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning due to a strong modulation of ventrolateral mesoderm patterning. Finally, a Cg-TGFbetaRI cytokine activity during immune regulation in C. gigas has been investigated by real-time PCR in haemocytes and mantle edge during an in vivo bacterial LPS challenge. One piece of evidence from this study suggests that the molecular mechanisms controlling mesodermal patterning and some immune regulations across all bilateria could be conserved through a functional TGF-beta s.s. pathway in lophotrochozoans.